Compositions and methods for inhibiting endogenous immunoglobulin genes and producing transgenic human idiotype antibodies

ABSTRACT

The invention relates to transgenic animals lacking endogenous Ig and capable of producing transgenic antibodies, as well as methods of making the same. The invention further relates to methods for producing transgenic antibodies in such animals, and transgenic antibodies so produced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/941,619 filed 1 Jun. 2007, and U.S. provisional patentapplication Ser. No. 61/044,324 filed 11 Apr. 2008, which areincorporated herein in their entirety by reference.

SUMMARY OF THE INVENTION

The invention relates to transgenic animals having one or moreinactivated endogenous immunoglobulin loci and methods for making thesame. The invention further relates to compositions and methods for theproduction of humanized and fully human antibodies using such transgenicanimals, and antibodies so produced.

BACKGROUND OF THE INVENTION

Antibodies are an important class of pharmaceutical products that havebeen successfully used in the treatment of various human diseases andconditions, including infectious diseases, cancer, allergic diseases,and graft-versus-host disease, as well as in the prevention oftransplant rejection.

One problem associated with the therapeutic application of non-humanimmunoglobulins is the potential immunogenicity of the same in humanpatients. In order to reduce the immunogenicity of such preparations,various strategies for the production of partially human (humanized) andfully human antibodies have been developed. The ability to producetransgenic antibodies having a human idiotype in non-human animals isparticularly desirable as antigen binding determinants lie within theidiotype region, and non-human idiotypes are thought to contribute tothe immunogenicity of current antibody therapeutics. Human idiotype isan especially important consideration in respect of monoclonal antibodytherapeutics, which consist of a single idiotype delivered at relativelyhigh concentration as opposed to the variety of idiotypes delivered atlower concentrations by a polyclonal antibody mixture.

While a number of approaches to producing humanized transgenicantibodies in non-human animals have been described, one major problemencountered in many such approaches is the production of endogenousantibody, either preferentially or in combination with transgenicantibodies in the host animal. Various recombinant cloning schemes havebeen used in attempts to disrupt endogenous immunoglobulin production inhost animals to address this problem. However, the functionalinactivation of immunoglobulin genes presents many obstacles in manyvertebrate species.

For example, while homozygous mutant mice with deleted JH-loci have beensuccessfully produced using homologous recombination, ES or othersustainable pluripotent cells in which homologous recombination can bedone to inactivate endogenous loci are not readily available from mostvertebrate species.

Further, mutations that interfere with cell surface expression but notwith productive rearrangement of immunoglobulin VDJ or VJ gene-segmentsare insufficient to inactivate endogenous Ig expression completely. Thisis exemplified by the fact that homozygous mutant mice with a disruptedmembrane exon of the μ heavy chain (so called μMT mice) cannot produceIgM or IgG, but still produce significant quantities of IgA (Macpehrsonet al. Nature Immunol 2(7):625-631 (2001). In addition, the serum ofheterozygous mutant mice contains IgM and IgG encoded by both alleles,the wild-type allele and the mutated μMT allele (Kitamura and Rajewky,Nature 356:154-156 (1992). This is due to the fact that the firstrearrangement in the course of B-cell development is the joining of DH-and JH-gene segments on both homologous chromosomes, generating a pro-Bcell. If, in the μMT/+ mice, a pro-B cell undergoes subsequent VH-DHJHjoining in the mutated IgH locus first and the joining is in frame(“productive”), the resulting pre-B cell can express a μ chain of thesecreted form, but cannot express membrane-bound μ. Since membrane-boundμ expression is required for allelic exclusion, such a cell is stillable to undergo VH-DHJH joining in the wild-type IgH locus; and if thissecond rearrangement is also productive, the cell expresses twodifferent μ chains, one of which is membrane-bound. Serum of such micecontains IgM derived from both alleles. In addition, IgG derived fromboth alleles can be found in the serum of such mice because switching isoften concomitantly induced on both IgH loci of a B cell.

Incomplete allelic exclusion is also observed in animals with functionaltransgenic immunoglobulin loci and mutated endogenous immunoglobulinloci that can still rearrange VDJ or VJ gene segments productively. AB-cell rearranging VH-DHJH in one or both mutated endogenous loci maystill rearrange transgenic immunoglobulin loci productively. Such aB-cell expresses membrane-bound transgenic immunoglobulin and developsinto a mature B-cell. During B-cell development isotype switching in themutated endogenous locus may result in a B-cell expressing endogenousimmunoglobulin. Accordingly, such mutations are insufficient for thecomplete inactivation of endogenous immunoglobulin expression in animalswith transgenic immunoglobulin loci.

SUMMARY OF THE INVENTION

A major problem associated with the production of humanized transgenicantibodies in non-human animals has been the preferential production orco-production of endogenous antibodies in the host. The currentinvention solves this problem by providing transgenic animals thatharbor at least one artificial Ig locus and lack the capacity to produceendogenous immunoglobulin. These animals are highly useful for theproduction of humanized and fully human transgenic antibodies. Themethods used to generate such transgenic animals are effective in manyspecies, including species from which ES cells or sustainablepluripotent cells are not currently readily available and in whichhomologous recombination and gene knockouts are not readily done.

The present invention stems in part from the finding that a meganucleasemay be used to functionally ablate endogenous immunoglobulin loci togenerate transgenic animals useful for the production of humanized andfully human transgenic antibodies. Further, two distinct meganucleasestargeting distinct genomic sites may be used to effectively delete alarge portion of an immunoglobulin locus (up to several kb), therebyensuring complete inactivation of the locus and further ensuring thattransgenic animals carrying the germline mutation do not generate any Bcells capable of endogenous immunoglobulin production.

Accordingly, in one aspect, the invention provides transgenic animalscomprising at least one artificial Ig locus and having at least onegermline inactivated endogenous Ig locus. The animals used in theinvention are small laboratory animals, particularly birds, rodents andweasels. The artificial loci used in the invention comprise at least onehuman V gene segment. In a preferred embodiment, an artificial Ig locuscomprises (i) a V-region having at least one human V gene segmentencoding a germline or hypermutated human V-region amino acid sequence;(ii) one or more J gene segments; and (iii) one or more constant regiongenes, wherein the artificial Ig locus is functional and capable ofundergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the transgenic animal.

In one embodiment, the transgenic animal comprises an inactivatedendogenous Ig heavy chain locus. In a preferred embodiment, thetransgenic animal has both endogenous Ig heavy chain loci inactivatedand accordingly does not carry a functional endogenous Ig heavy chainlocus.

In one embodiment, the transgenic animal comprises an inactivatedendogenous Ig light chain locus. In a preferred embodiment, thetransgenic animal has both endogenous Ig light chain loci inactivatedand accordingly does not carry a functional endogenous Ig light chainlocus.

In a preferred embodiment, the transgenic animal lacks a functionalendogenous Ig heavy chain locus and a functional Ig light chain locus.

In one embodiment, the transgenic animal comprises at least oneartificial Ig heavy chain locus. In one embodiment, the transgenicanimal lacks a functional Ig light chain locus and comprises at leastone artificial Ig heavy chain locus.

In one embodiment, the transgenic animal comprises at least oneartificial Ig light chain locus.

In one embodiment, the transgenic animal comprises at least oneartificial Ig heavy chain locus and at least one artificial Ig lightchain locus.

In a preferred embodiment, artificial Ig loci are functional and capableof undergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the transgenic animal, which repertoire ofimmunoglobulins includes immunoglobulins having a human idiotype.

In one embodiment, one or more constant region genes of the artificialIg loci comprise at least one non-human constant region gene and arefunctional and capable of undergoing gene rearrangement and producing arepertoire of chimeric immunoglobulins in the transgenic animal, whichrepertoire of chimeric immunoglobulins includes chimeric immunoglobulinshaving a human idiotype.

In one embodiment, one or more constant region genes of the artificialIg loci comprise at least one human constant region gene and arefunctional and capable of undergoing gene rearrangement and producing arepertoire of immunoglobulins in the transgenic animal, which repertoireof immunoglobulins includes immunoglobulins having a human idiotype andhuman constant region.

In one aspect, the invention provides descendants of transgenic animalsof the invention. In a preferred embodiment, descendants comprise atleast one artificial Ig locus and have at least one germline inactivatedendogenous Ig locus.

In one aspect, the invention provides transgenic animals capable ofgenerating viable germ cells having at least one endogenous Ig locusthat is inactivated.

In one embodiment, such transgenic animals comprise a genomicmeganuclease expression construct, preferably a construct having aninducible expression control region operably linked to ameganuclease-encoding nucleic acid, wherein the encoded meganucleaserecognizes a meganuclease target sequence present in or proximal to anendogenous Ig locus of the transgenic animal. When the transgenic animalis sexually mature and comprises viable germ cells, and the genomicmeganuclease expression construct may be used to inactivate the targetedendogenous Ig locus in such germ cells, in vitro or in vivo, withoutcompromising the viability thereof, ensuring F1 animals carrying agermline mutation in an Ig locus may be derived therefrom.

In one embodiment, the transgenic animal further comprises at least oneartificial Ig locus.

In one aspect, the invention provides transgenic animals comprisingviable germ cells wherein at least one endogenous Ig locus isinactivated. In one embodiment, the transgenic animal further comprisesat least one artificial Ig locus.

In one aspect, the invention provides methods for producing transgenicanimals of the invention.

In one embodiment, the invention provides methods for producingtransgenic animals comprising at least one artificial Ig locus andhaving at least one germline inactivated endogenous Ig locus. In apreferred embodiment, the transgenic animal is nullizygous forendogenous Ig light chain and/or endogenous Ig heavy chain.

Preferably, an endogenous Ig locus is inactivated in a parent germ cell,or the germ cell of a predecessor, by expression of a meganucleasetherein. The methods comprise producing a meganuclease in the germ cell,wherein the meganuclease recognizes a meganuclease target sequencepresent in or proximal to an endogenous Ig locus and selectivelyinactivates the targeted Ig locus in the germ cell thereby producing aviable germ cell having at least one inactivated endogenous Ig locus.Such a germ cell having at least one inactivated endogenous Ig locus isused to produce an animal having at least one germline inactivatedendogenous Ig locus. In one embodiment, the germ cell, or that which itis combined with, comprises at least one artificial Ig heavy chainlocus. In one embodiment, the germ cell, or that which it is combinedwith, comprises at least one artificial Ig light chain locus. In oneembodiment, the germ cell, or that which it is combined with, comprisesat least one artificial Ig light chain locus and at least one artificialIg heavy chain locus.

In one embodiment, the methods involve introducing a meganucleaseexpression construct or meganuclease-encoding nucleic acid into the germcell.

In a preferred embodiment, the germ cell comprises a genomicmeganuclease expression construct, which comprises an expression controlregion operably linked to a meganuclease-encoding nucleic acid. In apreferred embodiment, the germ cell comprises an inducible genomicmeganuclease expression construct and the methods involve inducingexpression of the meganuclease-encoding nucleic acid in the germ cell.In one embodiment, the methods involve repeating the step of inducingexpression of the meganuclease-encoding nucleic acid in the germ cell.In one embodiment, induction is done in vivo. In another embodiment,induction is done in vitro. In one embodiment, the germ cell comprises agenomic meganuclease expression construct, which comprises an expressioncontrol region that exhibits germ cell-specific activity.

Resultant germ cells may be used to generate an F1 animal having atleast one germline inactivated endogenous Ig locus. The F1 animal maycomprise one or more artificial Ig loci or may be crossed in order togenerate such animals comprising at least one artificial Ig locus.

In an alternative embodiment, the method involves introducing ameganuclease expression construct or meganuclease-encoding nucleic acidinto a fertilized oocyte or embryo and generating a viable germ cellhaving at least one inactivated Ig locus in the resultant founderanimal. The founder animal can be used to generate an F1 animal havingat least one germline inactivated endogenous Ig locus. The F1 animal maycomprise one or more artificial Ig loci or may be crossed in order togenerate such animals comprising at least one artificial Ig locus.

In one embodiment, the meganuclease target sequence is present in orproximal to a J gene segment.

In one embodiment, the meganuclease target sequence is present in orproximal to an immunoglobulin constant region gene segment. In apreferred embodiment, the constant region gene encodes immunoglobulin p.

In one embodiment, the methods involve screening germ cells forviability and inactivation of an endogenous Ig locus. In one embodiment,the methods involve screening germ cells for the presence of anartificial Ig locus.

In methods herein, the crossing of animals is preferably between animalshaving inactivated endogenous loci, to generate animals that arenullizygous for endogenous Ig light chain and/or endogenous Ig heavychain.

In a preferred embodiment, the methods further comprise the use of asecond meganuclease. The second meganuclease recognizes a secondmeganuclease target sequence present in or proximal to the endogenous Iglocus and selectively cleaves the endogenous Ig locus together with thefirst meganuclease but at a site distinct from that of the firstmeganuclease, thereby inactivating at least one endogenous Ig locus.

In a preferred embodiment, the germ cell comprises a second genomicmeganuclease expression construct, which comprises an expression controlregion operably linked to a second meganuclease-encoding nucleic acid.In a preferred embodiment, the expression control region is an inducibleexpression control region, and the method further comprises inducingexpression of the second meganuclease-encoding nucleic acid in the germcell, whereby the encoded second meganuclease is produced and, togetherwith the first meganuclease, selectively inactivates the targeted Iglocus in the germ cell. In one embodiment, the methods involve repeatingthe step of inducing expression of the second meganuclease-encodingnucleic acid in the germ cell. In one embodiment, induction is done invivo. In one embodiment, induction is done in vitro. In one embodiment,the second genomic meganuclease expression construct comprises anexpression control region that exhibits germ cell-specific activity.

In an alternative embodiment, the methods involve introducing a secondmeganuclease expression construct or second meganuclease-encodingnucleic acid into the germ cell.

In an alternative embodiment, the methods involve introducing a secondmeganuclease expression construct or second meganuclease-encodingnucleic acid into a fertilized oocyte or embryo and generating a viablegerm cell having at least one inactivated Ig locus in the resultantfounder animal. The founder animal can be used to generate an F1 animalhaving at least one germline inactivated endogenous Ig locus. The F1animal may comprise one or more artificial Ig loci or may be crossed inorder to generate such animals comprising at least one artificial Iglocus.

In a preferred embodiment, the first and second meganucleases target Jgene segments. In one embodiment, the first and second meganucleasetarget sequences are, taken together, upstream and downstream of one ormore J gene segments within the endogenous Ig locus, and cleavage by thefirst and second encoded meganucleases produces deletion of a genomicDNA segment comprising the one or more J gene segments.

In another embodiment, the first and second meganucleases targetconstant region gene segments. In one embodiment, the first and secondmeganuclease target sequences are, taken together, upstream anddownstream of one or more immunoglobulin constant region gene segments,and cleavage by the first and second encoded meganucleases producesdeletion of a genomic DNA segment comprising the one or moreimmunoglobulin constant region gene segments. In a preferred embodiment,the constant region gene encodes immunoglobulin p.

In methods herein, the artificial loci used comprise at least one humanV gene segment. In a preferred embodiment, an artificial Ig locuscomprises (i) a V-region having at least one human V gene segmentencoding a germline or hypermutated human V-region amino acid sequence;(ii) one or more J gene segments; and (iii) one or more constant regiongenes, wherein the artificial Ig locus is functional and capable ofundergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the transgenic animal.

In one embodiment, at least one artificial Ig heavy chain locus isincorporated into the genome of a transgenic animal of the invention. Inone embodiment, the transgenic animal lacks a functional Ig light chainlocus.

In one embodiment, at least one artificial Ig light chain locus isincorporated into the genome of a transgenic animal of the invention.

In one embodiment, at least one artificial Ig heavy chain locus and atleast one artificial Ig light chain locus are incorporated into thegenome of a transgenic animal of the invention.

In a preferred embodiment, artificial Ig loci are functional and capableof undergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the transgenic animal, which repertoire ofimmunoglobulins includes immunoglobulins having a human idiotype.

In one embodiment, one or more constant region genes of the artificialIg loci comprise at least one non-human constant region gene and arefunctional and capable of undergoing gene rearrangement and producing arepertoire of chimeric immunoglobulins in the transgenic animal, whichrepertoire of chimeric immunoglobulins includes chimeric immunoglobulinshaving a human idiotype.

In one embodiment, one or more constant region genes of the artificialIg loci comprise at least one human constant region gene and arefunctional and capable of undergoing gene rearrangement and producing arepertoire of immunoglobulins in the transgenic animal, which repertoireof immunoglobulins includes immunoglobulins having a human idiotype andhuman constant region.

In one embodiment, the methods of making a transgenic animal of theinvention comprise crossing a transgenic animal having at least onegermline inactivated endogenous Ig locus with a second transgenic animalhaving at least one artificial Ig locus, which locus comprises (i) aV-region having at least one human V gene segment encoding a germline orhypermutated human V-region amino acid sequence; (ii) one or more J genesegments; and (iii) one or more constant region genes, to produce an F1transgenic animal, wherein the F1 transgenic animal comprises the atleast one artificial Ig locus of the second transgenic animal, andwherein the artificial Ig locus from the second transgenic animal isfunctional and capable of undergoing gene rearrangement and producing arepertoire of immunoglobulins in the F1 transgenic animal. The crossingmay be done by animal breeding or by otherwise combining gametes,including in vitro manipulations.

In one embodiment, the second transgenic animal comprises at least oneartificial Ig heavy chain locus.

In one embodiment, the second transgenic animal comprises at least oneartificial Ig light chain locus.

In one embodiment, the first and second transgenic animals lack afunctional Ig light chain locus, and the second transgenic animalcomprises an artificial Ig heavy chain locus. The animals may be crossedto produce an F1 that lacks a functional Ig light chain locus andcomprises an artificial Ig heavy chain locus.

In one embodiment, the second transgenic animal comprises at least twoartificial Ig loci, including at least one artificial Ig heavy chainlocus and at least one artificial Ig light chain locus. In oneembodiment, the artificial Ig loci of the second transgenic animal arefunctional and capable of undergoing gene rearrangement and producing arepertoire of immunoglobulins in the F1 transgenic animal, whichrepertoire of immunoglobulins includes immunoglobulins having a humanidiotype. In one embodiment, one or more constant region genes of theartificial Ig loci of the second transgenic animal comprise at least onenon-human constant region gene and are functional and capable ofundergoing gene rearrangement and producing a repertoire of chimericimmunoglobulins in the F1 transgenic animal, which repertoire ofchimeric immunoglobulins includes chimeric immunoglobulins having ahuman idiotype. In one embodiment, one or more constant region genes ofthe artificial Ig loci of the second transgenic animal comprise at leastone human constant region gene and are functional and capable ofundergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the F1 transgenic animal, which repertoire ofimmunoglobulins includes immunoglobulins having a human idiotype andhuman constant region.

Similarly, in one embodiment, the methods comprise crossing a secondtransgenic animal having at least one artificial Ig locus with atransgenic animal of the invention that is capable of generating aviable germ cell having at least one endogenous Ig locus that isinactivated. In a preferred embodiment, the second transgenic animalcomprises at least two artificial Ig loci, including at least oneartificial Ig heavy chain locus and at least one artificial Ig lightchain locus.

In one embodiment, the methods comprise introducing at least oneartificial Ig locus into a germ cell having at least one endogenous Iglocus that has been, or is capable of being inactivated by the activityof one or more meganucleases, wherein the at least one artificial Iglocus comprises (i) a V-region having at least one human V gene segmentencoding a germline or hypermutated human V-region amino acid sequence;(ii) one or more J gene segments; and (iii) one or more constant regiongenes, wherein the artificial Ig locus is functional and capable ofundergoing gene rearrangement and producing a repertoire of artificialimmunoglobulins in a transgenic animal derived from the germ cell. Themethods further comprise deriving an F1 transgenic animal comprising atleast one artificial Ig locus and having at least one germlineinactivated endogenous Ig locus that has been inactivated by the actionof one or more meganucleases from the germ cell so produced.

In one embodiment, the at least one artificial Ig locus includes atleast one artificial Ig heavy chain locus.

In one embodiment, the germ cell lacks a functional Ig light chain locusand the artificial Ig locus introduced into the germ cell is an Ig heavychain locus.

In one embodiment, the at least one artificial Ig locus includes atleast one artificial Ig light chain locus.

In a preferred embodiment, at least two artificial loci are introducedinto the germ cell, including at least one artificial Ig heavy chainlocus and at least one artificial Ig light chain locus. In oneembodiment, the artificial Ig loci are functional and capable ofundergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the derived F1 transgenic animal, which repertoire ofimmunoglobulins includes immunoglobulins having a human idiotype. In oneembodiment, one or more constant region genes of the artificial Ig locicomprise at least one non-human constant region gene and are functionaland capable of undergoing gene rearrangement and producing a repertoireof chimeric immunoglobulins in the derived F1 transgenic animal, whichrepertoire of chimeric immunoglobulins includes chimeric immunoglobulinshaving a human idiotype. In one embodiment, one or more constant regiongenes of the artificial Ig loci comprise at least one human constantregion gene and are functional and capable of undergoing generearrangement and producing a repertoire of immunoglobulins in thederived F1 transgenic animal, which repertoire of immunoglobulinsincludes immunoglobulins having a human idiotype and human constantregion.

In one embodiment, the methods involve screening germ cells forviability and inactivation of an endogenous Ig locus. In one embodiment,the methods involve screening germ cells for the presence of anartificial Ig locus.

In one embodiment, the methods comprise introducing at least oneartificial Ig locus into a fertilized oocyte or embryo derived from agerm cell having at least one endogenous Ig locus that has beeninactivated, or is capable of being inactivated, by the action of one ormore meganucleases, wherein the at least one artificial Ig locuscomprises (i) a V-region having at least one human V gene segmentencoding a germline or hypermutated human V-region amino acid sequence;(ii) one or more J gene segments; and (iii) one or more constant regiongenes, wherein the artificial Ig locus is functional and capable ofundergoing gene rearrangement and producing a repertoire of artificialimmunoglobulins in the founder transgenic animal, or a descendantthereof, derived from the fertilized oocyte or embryo. The methodsfurther comprise deriving from the fertilized oocyte or embryo thefounder transgenic animal, and optionally the descendant thereof, toyield a transgenic animal comprising at least one artificial Ig locusand having at least one germline inactivated endogenous Ig locus thathas been inactivated by the action of one or more meganucleases.

In one embodiment, the at least one artificial Ig locus includes atleast one artificial Ig heavy chain locus.

In one embodiment, the at least one artificial Ig locus includes atleast one artificial Ig light chain locus.

In one embodiment, the fertilized oocyte or embryo lacks a functional Iglight chain locus, and the artificial Ig locus introduced into thefertilized oocyte or embryo is an Ig heavy chain locus.

In a preferred embodiment, at least two artificial loci are introducedinto the fertilized oocyte or embryo, including at least one artificialIg heavy chain locus and at least one artificial Ig light chain locus.In one embodiment, the artificial Ig loci are functional and capable ofundergoing gene rearrangement and producing a repertoire ofimmunoglobulins in the founder transgenic animal, or a descendantthereof, which repertoire of immunoglobulins includes immunoglobulinshaving a human idiotype. In one embodiment, one or more constant regiongenes of the artificial Ig loci comprise at least one non-human constantregion gene and are functional and capable of undergoing generearrangement and producing a repertoire of chimeric immunoglobulins inthe founder transgenic animal, or a descendant thereof, which repertoireof chimeric immunoglobulins includes chimeric immunoglobulins having ahuman idiotype. In one embodiment, one or more constant region genes ofthe artificial Ig loci comprise at least one human constant region geneand are functional and capable of undergoing gene rearrangement andproducing a repertoire of immunoglobulins in the founder transgenicanimal, or a descendant thereof, which repertoire of immunoglobulinsincludes immunoglobulins having a human idiotype and human constantregion.

In one aspect, the invention provides methods for producing transgenicanimals capable of generating a viable germ cell wherein at least oneendogenous Ig locus is inactivated. In a preferred embodiment, themethods comprise generating a transgenic animal having a genomicmeganuclease expression construct, wherein the expression constructcomprises an expression control region operably linked to ameganuclease-encoding nucleic acid. In a preferred embodiment, theconstruct is an inducible genomic meganuclease expression construct thatcan be induced to express the meganuclease-encoding nucleic acid in agerm cell

In one aspect, the invention provides methods for producing a transgenicanimal having a viable germ cell wherein at least one endogenous Iglocus is inactivated. The methods comprise inactivating the endogenousIg locus in the germ cell, or in a parent germ cell or fertilized oocyteor embryo derived therefrom, by expression of a meganuclease therein.

In one aspect, the invention provides a viable germ cell wherein atleast one endogenous Ig locus is capable of being inactivated. In apreferred embodiment, the germ cell comprises a genomic meganucleaseexpression construct, wherein the expression construct comprises anexpression control region operably linked to a meganuclease-encodingnucleic acid. In a preferred embodiment, the construct is an induciblegenomic meganuclease expression construct that can be induced to expressthe meganuclease-encoding nucleic acid in a germ cell.

In one embodiment, the germ cell comprises at least one artificial Igheavy chain locus.

In one embodiment, the germ cell comprises at least one artificial Iglight chain locus.

In one embodiment, the germ cell comprises at least one artificial Igheavy chain locus and at least one artificial Ig light chain locus.

In one aspect, the invention provides a viable germ cell wherein atleast one endogenous Ig locus is inactivated.

In one embodiment, the germ cell comprises at least one artificial Igheavy chain locus.

In one embodiment, the germ cell comprises at least one artificial Iglight chain locus.

In one embodiment, the germ cell comprises at least one artificial Igheavy chain locus and at least one artificial Ig light chain locus.

In one aspect, the invention provides methods for producing a viablegerm cell having at least one inactivated endogenous Ig locus. Themethods involve expressing at least one meganuclease in a germ cell,fertilized oocyte or embryo, to generate a viable germ cell having atleast one inactivated endogenous Ig locus. The meganuclease so expressedrecognizes a meganuclease target sequence present in or proximal to saidendogenous Ig locus.

In one embodiment, wherein the meganuclease is expressed in a germ cell,the germ cell in which the meganuclease is expressed yields a viablegerm cell having at least one inactivated endogenous Ig locus.Alternatively, a viable germ cell having at least one inactivatedendogenous Ig locus may be obtained from an animal derived from the germcell in which the meganuclease was expressed.

In one embodiment, wherein the meganuclease is expressed in a fertilizedoocyte or embryo, the viable germ cell having at least one inactivatedendogenous Ig locus may be obtained from an animal derived from thefertilized oocyte or embryo in which the meganuclease was expressed.

In one embodiment, the at least one endogenous Ig locus is inactivatedin vitro. In one embodiment, the at least one endogenous Ig locus isinactivated in vivo.

In one embodiment, the germ cell further comprises at least oneartificial Ig locus. In one embodiment, the at least one artificial Iglocus includes at least one artificial Ig heavy chain locus. In oneembodiment, the at least one artificial Ig locus includes at least oneartificial Ig light chain locus.

In one embodiment, at least two artificial Ig loci are introduced intothe germ cell, including at least one artificial Ig heavy chain locusand at least one artificial Ig light chain locus.

The invention also provides polyclonal antibodies, monoclonalantibodies, hybridomas, and methods of making and using the same, whichstem from the production of antibodies in the presently disclosedtransgenic animals carrying one or more artificial loci and having oneor more endogenous Ig loci inactivated by way of meganuclease activity.

In one embodiment, the antibodies are heavy chain-only antibodies, whichare produced using transgenic animals which lack a functional Ig lightchain locus and comprise an artificial heavy chain locus, achieved bymethods described herein.

In one aspect, the invention provides methods for producing antibodiesusing transgenic animals provided herein. The methods compriseimmunizing a transgenic animal of the invention, which animal has atleast one inactivated endogenous Ig locus and carries at least oneartificial Ig locus as described herein, with an immunogen. In apreferred embodiment, the transgenic animal is nullizygous forendogenous Ig heavy chain and/or endogenous Ig light chain and,accordingly, incapable of producing endogenous immunoglobulins. In oneembodiment, the transgenic animal lacks a functional Ig light chainlocus and comprises an artificial Ig heavy chain locus.

In one aspect, the invention provides polyclonal antisera compositionsso produced. Polyclonal antisera of the invention preferably compriseantibodies having a human idiotype. In a preferred embodiment, apolyclonal antiserum comprises antibodies that consist essentially ofantibodies having a human idiotype.

In one aspect, the invention provides methods for producing monoclonalantibodies.

In one embodiment, the methods comprise (i) immunizing a transgenicanimal of the invention, which animal has at least one inactivatedendogenous Ig locus and carries at least one artificial Ig locus asdescribed herein, with an immunogen; (ii) isolating a monoclonalantibody producing cell from the transgenic animal wherein themonoclonal antibody producing cell produces a monoclonal antibody thatspecifically binds to the immunogen; and (iii) using the monoclonalantibody producing cell to produce the monoclonal antibody thatspecifically binds to the immunogen, or using the monoclonal antibodyproducing cell to produce a hybridoma cell that produces the monoclonalantibody and using the hybridoma cell to produce the monoclonalantibody.

In one embodiment, the methods comprise (i) immunizing a transgenicanimal of the invention, which animal has at least one inactivatedendogenous Ig locus and carries at least one artificial Ig locus asdescribed herein, with an immunogen; (ii) isolating a monoclonalantibody producing cell from the transgenic animal wherein themonoclonal antibody producing cell produces a monoclonal antibody thatspecifically binds to the immunogen; (iii) isolating from the monoclonalantibody producing cell a monoclonal antibody nucleic acid which encodesthe monoclonal antibody that specifically binds to the immunogen; and(iv) using the monoclonal antibody nucleic acid to produce themonoclonal antibody that specifically binds to the immunogen.

In a preferred embodiment, the monoclonal antibody has a human idiotype.

In one aspect, the invention provides monoclonal antibodies so produced.

In one aspect, the invention provides isolated nucleic acids encodingsuch monoclonal antibodies.

In one aspect, the invention provides methods for producing fully humanmonoclonal antibodies. The methods comprise (i) immunizing a transgenicanimal of the invention, which animal has at least one inactivatedendogenous Ig locus and carries at least one artificial Ig locus asdescribed herein, with an immunogen; (ii) isolating a monoclonalantibody producing cell from the transgenic animal wherein themonoclonal antibody producing cell produces a monoclonal antibody thatspecifically binds to the immunogen; (iii) isolating from the monoclonalantibody producing cell a monoclonal antibody nucleic acid which encodesthe monoclonal antibody that specifically binds to the immunogen; (iv)modifying the monoclonal antibody nucleic acid to produce a recombinantnucleic acid encoding a fully human monoclonal antibody; and (v) usingthe recombinant nucleic acid encoding a fully human monoclonal antibodyto produce the encoded fully human monoclonal antibody.

In one aspect, the invention provides fully human monoclonal antibodiesso produced.

In one aspect, the invention provides recombinant nucleic acids encodingfully human monoclonal antibodies, and methods of producing the same.

In one embodiment, an immunogen used in methods herein comprises adisease-causing organism or antigenic portion thereof.

In one embodiment, an immunogen used in methods herein is an antigenendogenous to humans. In an alternative embodiment, an immunogen used inmethods herein is an antigen exogenous to humans.

In one aspect, the invention provides methods for neutralizing ormodulating the activity of an antigenic entity in a human bodycomponent. In one embodiment, the methods comprise contacting the bodycomponent with a polyclonal antisera composition of the invention,wherein the polyclonal antisera composition comprises immunoglobulinmolecules that specifically bind to and neutralize or modulate theactivity of the antigenic entity.

In one embodiment, the methods comprise contacting the body componentwith a monoclonal antibody of the invention, wherein the monoclonalantibody specifically binds to and neutralizes or modulates the activityof the antigenic entity.

In a preferred embodiment, the monoclonal antibody is a fully humanmonoclonal antibody.

In one embodiment, the antigenic entity is from an organism that causesan infectious disease.

In one embodiment, the antigenic entity is a cell surface molecule.

In one embodiment, the antigenic entity is a human cytokine or a humanchemokine.

In one embodiment, the antigenic entity is a cell surface molecule on amalignant cancer cell.

In one aspect, the invention provides cells derived from transgenicanimals of the invention.

In a preferred embodiment, the invention provides cells derived from thespleen of transgenic animals of the invention.

In a preferred embodiment, the invention provides B cells derived fromtransgenic animals of the invention, which B cells are capable ofproducing antibodies having a human idiotype.

In a preferred embodiment, the invention provides germ cells derivedfrom transgenic animals of the invention.

In one aspect, the invention provides methods for making hybridomascapable of producing antibodies having a human idiotype. The methodscomprise the use of cells derived from transgenic animals of theinvention.

In one aspect, the invention provides hybridomas so produced.

In one aspect, the invention provides antibodies having a humanidiotype, which antibodies are produced by a hybridoma of the invention.

In one aspect, the invention provides pharmaceutical compositionscomprising an antibody of the invention, which antibody has a humanidiotype.

In one aspect, the invention provides methods of treating a patient inneed of treatment, comprising administering a therapeutically effectiveamount of a pharmaceutical composition of the invention to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of an artificial heavy chainconsisting of a human V-, D, and J-region, a rat intronic enhancer andseveral artificial constant region genes. Artificial constant regiongenes contain exons encoding a human CH1 domain and rat CH2,3 and 4domains. Membrane spanning and cytoplasmic polypeptide sequences areencoded by rat exons.

FIG. 2. Schematic of the interaction of I-SceI and DNA at 3′ end ofrecognition sequence.

FIG. 3. Schematic of the interaction of the 5′ end of the I-SceIrecognition sequence with I-SceI.

FIG. 4. Schematic of sequence recognition mechanism of I-CreI (fromNucleic Acids Res., 34, 4791-4800).

FIG. 5. Schematic diagram of the strategy for altering recognitionsequence of I-CreI.

FIG. 6. Zinc-finger proteins (ZFP) designed against sequences encodingrat IgM were expressed in cells, chromosomal DNA was prepared, and theappropriate region of the IgM locus was PCR amplified. Reaction productswere analyzed by polyacrylamide gel electrophoresis. The figure shows atypical example demonstrating cleavage activity.

DETAILED DESCRIPTION OF THE INVENTION

By “artificial immunoglobulin locus” is meant an immunoglobulin locuscomprising fragments of human and non-human immunoglobulin loci,including multiple immunoglobulin gene segments, which include at leastone variable region (V) gene segment, one or more J gene segments, oneor more D gene segments in the case of a heavy chain locus, and one ormore constant region gene segments. In the present invention, at leastone of the V gene segments encodes a germline or hypermutated humanV-region amino acid sequence. In a preferred embodiment, an artificialimmunoglobulin locus of the invention is functional and capable ofrearrangement and producing a repertoire of immunoglobulins. In apreferred embodiment, at least one D gene segment is a human D genesegment. “Artificial Ig locus” as used herein can refer to unrearrangedloci, partially rearranged loci, and rearranged loci. Artificial Ig lociinclude artificial Ig light chain loci and artificial Ig heavy chainloci. In one embodiment, an artificial Ig locus comprises a non-human Cregion gene and is capable of producing a repertoire of immunoglobulinsincluding chimeric immunoglobulins having a non-human C region. In oneembodiment, an artificial Ig locus comprises a human C region gene andis capable of producing a repertoire of immunoglobulins includingimmunoglobulins having a human C region. In one embodiment, anartificial Ig locus comprises an “artificial constant region gene”, bywhich is meant a constant region gene comprising nucleotide sequencesderived from human and non-human constant regions genes. For example, anexemplary artificial C constant region gene is a constant region geneencoding a human IgG CH1 domain and rat IgG CH2 and CH3 domain.

In some embodiments, an artificial Ig heavy chain locus lacks CH1, or anequivalent sequence that allows the resultant immunoglobulin tocircumvent the typical immunoglobulin: chaperone association. Suchartificial loci provide for the production of heavy chain-onlyantibodies in transgenic animals which lack a functional Ig light chainlocus and hence do not express functional Ig light chain. Suchartificial Ig heavy chain loci are used in methods herein to producetransgenic animals lacking a functional Ig light chain locus, andcomprising an artificial Ig heavy chain locus, which animals are capableof producing heavy chain-only antibodies. Alternatively, an artificialIg locus may be manipulated in situ to disrupt CH1 or an equivalentregion and generate an artificial Ig heavy chain locus that provides forthe production of heavy chain-only antibodies. Regarding the productionof heavy chain-only antibodies in light chain-deficient mice, see forexample Zou et al., JEM, 204:3271-3283, 2007.

By “human idiotype” is meant a polypeptide sequence present on a humanantibody encoded by an immunoglobulin V-gene segment. The term “humanidiotype” as used herein includes both naturally occurring sequences ofa human antibody, as well as synthetic sequences substantially identicalto the polypeptide found in naturally occurring human antibodies. By“substantially” is meant that the degree of amino acid sequence identityis at least about 85%-95%. Preferably, the degree of amino acid sequenceidentity is greater than 90%, more preferably greater than 95%.

By a “chimeric antibody” or a “chimeric immunoglobulin” is meant animmunoglobulin molecule comprising a portion of a human immunoglobulinpolypeptide sequence (or a polypeptide sequence encoded by a human Iggene segment) and a portion of a non-human immunoglobulin polypeptidesequence. The chimeric immunoglobulin molecules of the present inventionare immunoglobulins with non-human Fc-regions or artificial Fc-regions,and human idiotypes. Such immunoglobulins can be isolated from animalsof the invention that have been engineered to produce chimericimmunoglobulin molecules.

By “artificial Fc-region” is meant an Fc-region encoded by an artificialconstant region gene.

The term “Ig gene segment” as used herein refers to segments of DNAencoding various portions of an Ig molecule, which are present in thegermline of non-human animals and humans, and which are brought togetherin B cells to form rearranged Ig genes. Thus, Ig gene segments as usedherein include V gene segments, D gene segments, J gene segments and Cregion gene segments.

The term “human Ig gene segment” as used herein includes both naturallyoccurring sequences of a human Ig gene segment, degenerate forms ofnaturally occurring sequences of a human Ig gene segment, as well assynthetic sequences that encode a polypeptide sequence substantiallyidentical to the polypeptide encoded by a naturally occurring sequenceof a human Ig gene segment. By “substantially” is meant that the degreeof amino acid sequence identity is at least about 85%-95%. Preferably,the degree of amino acid sequence identity is greater than 90%, morepreferably greater than 95%.

By “meganuclease” is meant an endodeoxyribonuclease that recognizes longrecognition sites in DNA, preferably at least 12, more preferably atleast 13, more preferably at least 14, more preferably at least 15, morepreferably at least 16, more preferably at least 17, and most preferablyat least 18 nucleotides in length. Meganucleases include zinc-fingernucleases, naturally occurring homing endonucleases and customengineered zinc-finger nucleases and homing endonucleases. What isrequired for use in the invention is that the meganuclease recognize ameganuclease target sequence present in or proximal to an endogenous Iglocus in the subject animal such that a functional mutation may beintroduced in the Ig locus by the action of the meganuclease. For morediscussion of meganucleases, see, for example, U.S. Patent ApplicationPublication Nos. 20060206949, 20060153826, 20040002092, 20060078552, and20050064474.

Zinc-finger nucleases with altered specificity can be generated bycombining individual zinc fingers with different triplet targets. Thespecificity of naturally occurring homing endonucleases can be alteredby structure-based protein engineering. For example, see Proteus andCarroll, nature biotechnology 23(8):967-97, 2005.

An animal having a “germline inactivated Ig locus”, or “germlineinactivated endogenous Ig locus”, or “germline mutation in an endogenousIg locus”, has an inactivated endogenous Ig locus in every cell, i.e.,every somatic and germ cell. In the present invention, animals havinggermline inactivated loci are produced by mutation, as effected by theaction of a meganuclease in a germ cell which gives rise to theresultant animal, or a predecessor thereof.

Production of Viable Germ Cells and Transgenic Animals HavingInactivated Endogenous Ig Loci

In the present invention, meganucleases are used to inactivateendogenous Ig loci so as to produce viable germ cells having at leastone inactivated endogenous Ig locus. The methods involve expressing atleast one meganuclease in a germ cell, fertilized oocyte or embryo, togenerate a viable germ cell having at least one inactivated endogenousIg locus. The meganuclease so expressed recognizes a meganuclease targetsequence present in or proximal to an endogenous Ig locus in the subjectanimal.

In one embodiment, wherein the meganuclease is expressed in a germ cell,the germ cell in which the meganuclease is expressed yields a viablegerm cell having at least one inactivated endogenous Ig locus.Alternatively, a viable germ cell having at least one inactivatedendogenous Ig locus may be obtained from an animal derived from the germcell in which the meganuclease was expressed.

In one embodiment, wherein the meganuclease is expressed in a fertilizedoocyte or embryo, the viable germ cell having at least one inactivatedendogenous Ig locus may be obtained from an animal derived from thefertilized oocyte or embryo in which the meganuclease was expressed.

The invention also provides methods for producing transgenic animalscomprising at least one germline inactivated endogenous Ig locus. Themethods comprise deriving a transgenic animal from a viable germ cellhaving at least one inactivated endogenous Ig locus produced accordingto the methods herein.

In one embodiment, the viable germ cell having at least one inactivatedendogenous Ig locus further comprises an artificial Ig locus, and thetransgenic animal so produced comprises an artificial Ig locus.

In one embodiment, the methods further comprise introducing anartificial Ig locus into the viable germ cell having at least oneinactivated endogenous Ig locus, or a germ cell descendant thereof or afertilized oocyte or embryo derived therefrom, and the transgenic animalso produced comprises an artificial Ig locus.

In one embodiment, the methods comprise combining a viable germ cellhaving at least one inactivated endogenous Ig locus, or a germ celldescendant thereof, with a gamete comprising an artificial Ig locus, andthe transgenic animal so produced comprises an artificial Ig locus.

Inactivation of Endogenous Ig Loci

Inactivation of endogenous Ig loci is done using meganucleases specificfor immunoglobulin gene fragments in heavy and/or light chain lociendogenous to the subject animal. In one embodiment double-strand breaksmay be induced by injection of a meganuclease into germ cells,fertilized oocytes or embryos. Alternatively, expression vectors ornucleic acid encoding a meganuclease and capable of being expressed ingerm cells, fertilized oocytes or embryos may be injected into the same.

In one embodiment, the method involves transfecting germ cells, whichmay include precursors thereof such as spermatagonial stem cells, invitro or in vivo with a meganuclease encoding nucleic acid or expressionconstruct. For example, see Ryu et al., J. Androl., 28:353-360, 2007;Orwig et al., Biol. Report, 67:874-879, 2002.

In a preferred embodiment, a meganuclease expression construct isintegrated into the genome of the subject animal. Expression of thetransgene encoding the meganuclease in germ cells will result indouble-strand breaks in endogenous Ig loci and subsequent mutation ofthe restriction site. Mating of such transgenic animals results inoffspring with mutated/inactivated immunoglobulin loci.

In a highly preferred embodiment of the present invention, a regulatablemeganuclease expression construct is integrated into the genome of thesubject animal, which regulatable construct is inducible in germ cells.Such constructs provide for minimization of cytotoxic effects associatedwith expression of a particular meganuclease through controlledexpression via inducible promoters, e.g., heat-inducible promoters,radiation-inducible promoters, tetracycline operon, hormone induciblepromoters, and promoters inducible by dimerization of transactivators,and the like. For example, see Vilaboa et al., Current Gene Therapy,6:421-438, 2006.

Alternatively, meganuclease expression may be induced in an embryoderived from the germ cell.

In one embodiment, a single meganuclease is expressed in a germ cell,wherein the meganuclease recognizes a target sequence in or proximal toan immunoglobulin locus endogenous to the germ cell of the subjectanimal. In a preferred embodiment, the meganuclease target sequence isin or proximal to a J gene segment. In another preferred embodiment, themeganuclease target sequence is in or proximal to an immunoglobulinconstant region gene. In a preferred embodiment, the immunoglobulinconstant region gene encodes immunoglobulin p.

In a preferred embodiment, at least two meganucleases having distincttarget sequences are used. The at least two meganucleases are expressedin a germ cell, wherein the meganucleases recognize distinct targetsequences in or proximal to an immunoglobulin locus endogenous to thegerm cell of the subject animal.

In a preferred embodiment, the first and second meganucleases target Jgene segments. In one embodiment, the first and second meganucleasetarget sequences are, taken together, upstream and downstream of one ormore J gene segments within the endogenous Ig locus, and cleavage by thefirst and second encoded meganucleases produces deletion of a genomicDNA segment comprising the one or more J gene segments.

In another embodiment, the first and second meganucleases targetconstant region gene segments. In one embodiment, the first and secondmeganuclease target sequences are, taken together, upstream anddownstream of one or more immunoglobulin constant region gene segments,and cleavage by the first and second encoded meganucleases producesdeletion of a genomic DNA segment comprising the one or moreimmunoglobulin constant region gene segments. In a preferred embodiment,the constant region gene encodes immunoglobulin p.

In one embodiment, an entire endogenous Ig heavy chain and/or Ig lightchain locus, or large parts thereof are deleted from the genome of thesubject animal. Such animals are also referred to as comprising anendogenous locus that has been inactivated.

In one embodiment, at least one meganuclease is used to disrupt the CH1region of an endogenous Ig heavy chain locus, leaving the remainder ofthe locus intact and capable of producing an Ig heavy chain thatcircumvents the typical immunoglobulin:chaperone association.Preferably, this CH1 targeting is done in an animal lacking a functionalIg light chain locus. Such targeting in such animals is useful forproducing heavy chain-only antibodies.

In one embodiment, more than one meganuclease is used to target CH1within the Ig heavy chain locus.

In one embodiment, two meganucleases recognizing adjacent sites areused. In one embodiment, the sites are elements of a palindrome. In oneembodiment, the two meganucleases are tethered by a linker.

In preferred embodiments, the breeding strategies used are designed toobtain animals that are nullizygous for endogenous Ig light chain and/orendogenous Ig heavy chain.

Transgenic Animals Comprising Regulatable Genomic MeganucleaseExpression Constructs

In one aspect, the invention provides transgenic animals comprising atleast one regulatable genomic meganuclease expression construct.

The transgenic animals are selected from small laboratory animals,particularly birds (chicken, turkey, quail, duck, pheasant or goose andthe like), rodents (e.g., rats, hamsters and guinea pigs), and weasels(e.g., ferrets).

In a preferred embodiment, the regulatable genomic meganucleaseexpression construct comprises an inducible expression control regionoperably linked to a meganuclease-encoding nucleic acid. The inducibleexpression control region is inducibly functional in a germ cell of theparticular transgenic animal, and the encoded meganuclease is selectivefor a meganuclease target sequence situated in or proximal to anendogenous immunoglobulin locus of the subject animal.

A regulatable meganuclease expression construct provides forminimization of cytotoxic effects associated with expression of aparticular meganuclease through controlled expression via induciblepromoters, e.g., heat-inducible promoters, radiation-induciblepromoters, tetracycline operon, hormone inducible promoters, andpromoters inducible by dimerization of transactivators, and the like.

In a preferred embodiment, a transgenic animal of the inventioncomprises two regulatable genomic meganuclease expression constructs,comprising two distinct nucleic acids encoding two distinctmeganucleases that recognize two distinct target sequences. The twomeganucleases in combination function to delete a genomic DNA segment ofan endogenous Ig locus and thereby inactivate the same.

Transgenic animals comprising at least one regulatable genomicmeganuclease expression construct may be made by means well known in theart. For example, a transgenic vector containing an inducible expressioncontrol region operably linked to a meganuclease-encoding nucleic acidmay be introduced into a recipient cell or cells and then integratedinto the genome of the recipient cell or cells by random integration orby targeted integration.

For random integration, such a transgenic vector can be introduced intoa recipient cell by standard transgenic technology. For example, atransgenic vector can be directly injected into the pronucleus of afertilized oocyte. A transgenic vector can also be introduced byco-incubation of sperm with the transgenic vector before fertilizationof the oocyte. Transgenic animals can be developed from fertilizedoocytes. Another way to introduce a transgenic vector is by transfectingembryonic stem cells or other pluripotent cells (for example primordialgerm cells) and subsequently injecting the genetically modified cellsinto developing embryos. Alternatively, a transgenic vector (naked or incombination with facilitating reagents) can be directly injected into adeveloping embryo. In another embodiment, the transgenic vector isintroduced into the genome of a cell and an animal is derived from thetransfected cell by nuclear transfer cloning.

For targeted integration, such a transgenic vector can be introducedinto appropriate recipient cells such as embryonic stem cells or alreadydifferentiated somatic cells. Afterwards, cells in which the transgenehas integrated into the animal genome at the targeted site by homologousrecombination can be selected by standard methods. The selected cellsmay then be fused with enucleated nuclear transfer unit cells, e.g.oocytes or embryonic stem cells, cells which are totipotent and capableof forming a functional neonate. Fusion is performed in accordance withconventional techniques which are well established. See, for example,Cibelli et al., Science (1998) 280:1256 Zhou et al. Science (2003) 301:1179. Enucleation of oocytes and nuclear transfer can also be performedby microsurgery using injection pipettes. (See, for example, Wakayama etal., Nature (1998) 394:369.) The resulting cells are then cultivated inan appropriate medium, and transferred into synchronized recipients forgenerating transgenic animals. Alternatively, the selected geneticallymodified cells can be injected into developing embryos.

In one embodiment, a meganuclease is used to increase the frequency ofhomologous recombination at a target site through double-strand DNAcleavage.

Transgenic Animals Comprising Artificial Ig Loci and Capable ofProducing Antibodies Having Human Idiotypes

In one aspect, the invention provides transgenic animals capable ofproducing immunoglobulins having human idiotypes, as well as methods ofmaking the same.

The transgenic animals used are selected from particularly birds(chicken, turkey, qail, duck, pheasant or goose and the like), rodents(e.g., rats, hamsters and guinea pigs), and weasels (e.g., ferrets).

The transgenic animals used for humanized antibody production in theinvention carry germline mutations in endogenous Ig loci that have beeneffected by the activity of one or more meganucelases. In a preferredembodiment, the transgenic animals are nullizygous for endogenous Igheavy chain and/or endogenous Ig light chain. Further, these animalscarry at least one artificial Ig locus that is functional and capable ofproducing a repertoire of immunoglobulin molecules in the transgenicanimal. The artificial Ig loci used in the invention include at leastone human V gene segment.

In a preferred embodiment, the transgenic animals carry at least oneartificial Ig heavy chain locus and at least one artificial Ig lightchain locus that are each functional and capable of producing arepertoire of immunoglobulin molecules in the transgenic animal, whichrepertoire of immunoglobulin molecules includes antibodies having ahuman idiotype. In one embodiment, artificial loci including at leastone non-human C gene are used, and animals capable of producing chimericantibodies having a human idiotype and non-human constant region areprovided. In one embodiment, artificial loci including at least onehuman C gene are used, and animals capable of producing antibodieshaving a human idiotype and human constant region are provided.

In another preferred embodiment, the transgenic animals carry at leastone artificial Ig heavy chain locus, and lack a functional Ig lightchain locus. Such animals find use in the production of heavy chain—onlyantibodies.

Production of such transgenic animals involves the integration of one ormore artificial heavy chain Ig loci and one or more artificial lightchain Ig loci into the genome of a transgenic animal having at least oneendogenous Ig locus that has been or will be inactivated by the actionof one or more meganucleases. Preferably, the transgenic animals arenullizygous for endogenous Ig heavy chain and/or endogenous Ig lightchain and, accordingly, incapable of producing endogenousimmunoglobulins. Regardless of the chromosomal location, an artificialIg locus of the present invention has the capacity to undergo generearrangement and thereby produce a diversified repertoire ofimmunoglobulin molecules. An Ig locus having the capacity to undergogene rearrangement is also referred to herein as a “functional” Iglocus, and the antibodies with a diversity generated by a functional Iglocus are also referred to herein as “functional” antibodies or a“functional” repertoire of antibodies.

The artificial loci used to generate such transgenic animals eachinclude multiple immunoglobulin gene segments, which include at leastone V region gene segment, one or more J gene segments, one or more Dgene segments in the case of a heavy chain locus, and one or moreconstant region genes. In the present invention, at least one of the Vgene segments encodes a germline or hypermutated human V-region aminoacid sequence. Accordingly, such transgenic animals have the capacity toproduce a diversified repertoire of immunoglobulin molecules, whichinclude antibodies having a human idiotype.

In one embodiment, the artificial loci used comprise at least onenon-human C region gene segment. Accordingly, such transgenic animalshave the capacity to produce a diversified repertoire of immunoglobulinmolecules, which include chimeric antibodies having a human idiotype.

In one embodiment, the artificial loci used comprise at least one humanC region gene segment. Accordingly, such transgenic animals have thecapacity to produce a diversified repertoire of immunoglobulinmolecules, which include antibodies having a human idiotype and a humanconstant region.

In one embodiment, the artificial loci used comprise at least oneartificial constant region gene. For example, an exemplary artificial Cconstant region gene is a constant region gene encoding a human IgG CH1domain and rat IgG CH2 and CH3 domain. Accordingly, such transgenicanimals have the capacity to produce a diversified repertoire ofimmunoglobulin molecules, which include antibodies having a humanidiotype and an artificial constant region comprising both human andnon-human components.

The transgenic vector containing an artificial Ig locus is introducedinto the recipient cell or cells and then integrated into the genome ofthe recipient cell or cells by random integration or by targetedintegration.

For random integration, a transgenic vector containing an artificial Iglocus can be introduced into a recipient cell by standard transgenictechnology. For example, a transgenic vector can be directly injectedinto the pronucleus of a fertilized oocyte. A transgenic vector can alsobe introduced by co-incubation of sperm with the transgenic vectorbefore fertilization of the oocyte. Transgenic animals can be developedfrom fertilized oocytes. Another way to introduce a transgenic vector isby transfecting embryonic stem cells or other pluripotent cells (forexample primordial germ cells) and subsequently injecting thegenetically modified cells into developing embryos. Alternatively, atransgenic vector (naked or in combination with facilitating reagents)can be directly injected into a developing embryo. Ultimately, chimerictransgenic animals are produced from the embryos which contain theartificial Ig transgene integrated in the genome of at least somesomatic cells of the transgenic animal. In another embodiment, thetransgenic vector is introduced into the genome of a cell and an animalis derived from the transfected cell by nuclear transfer cloning.

In a preferred embodiment, a transgene containing an artificial Ig locusis randomly integrated into the genome of recipient cells (such asfertilized oocyte or developing embryos). The recipient cells arederived from an animal having at least one endogenous Ig locus that hasbeen inactivated by the action of one or more meganucleases.Alternatively, transgenic animals carrying artificial immunoglobulinloci, can be crossed with transgenic animals having at least oneendogenous Ig locus that has been inactivated by the action of one ormore meganucleases. Regardless of the particular method used, in apreferred embodiment, offspring that are nullizygous for endogenous Igheavy chain and/or Ig light chain and, accordingly, incapable ofproducing endogenous immunoglobulins and capable of producing transgenicimmunoglobulins are obtained.

For targeted integration, a transgenic vector can be introduced intoappropriate recipient cells such as embryonic stem cells, otherpluripotent cells or already differentiated somatic cells. Afterwards,cells in which the transgene has integrated into the animal genome andhas replaced the corresponding endogenous Ig locus by homologousrecombination can be selected by standard methods. The selected cellsmay then be fused with enucleated nuclear transfer unit cells, e.g.oocytes or embryonic stem cells, cells which are totipotent and capableof forming a functional neonate. Fusion is performed in accordance withconventional techniques which are well established. See, for example,Cibelli et al., Science (1998) 280:1256; Zhou et al. Science (2003) 301:1179. Enucleation of oocytes and nuclear transfer can also be performedby microsurgery using injection pipettes. (See, for example, Wakayama etal., Nature (1998) 394:369.) The resulting cells are then cultivated inan appropriate medium, and transferred into synchronized recipients forgenerating transgenic animals. Alternatively, the selected geneticallymodified cells can be injected into developing embryos which aresubsequently developed into chimeric animals.

In one embodiment, a meganuclease is used to increase the frequency ofhomologous recombination at a target site through double-strand DNAcleavage. For integration into endogenous immunoglobulin loci a sitespecific meganuclease may be used. In one embodiment, a meganucleasetargeting an endogenous Ig locus is used to increase the frequency ofhomologous recombination and replacement of an endogenous Ig locus, orparts thereof with an artificial Ig locus, or parts thereof.

In one embodiment, the transgenic animal lacks a functional Ig lightchain locus and comprises an artificial Ig heavy chain locus.

Artificial Ig Loci

The present invention is further directed to artificial Ig loci andtheir use in making transgenic animals capable of producingimmunoglobulins having a human idiotype.

Each artificial Ig locus comprises multiple immunoglobulin genesegments, which include at least one V region gene segment, one or moreJ gene segments, one or more D gene segments in the case of a heavychain locus, and one or more constant region genes. In the presentinvention, at least one of the V gene segments encodes a germline orhypermutated human V-region amino acid sequence. Accordingly, suchtransgenic animals have the capacity to produce a diversified repertoireof immunoglobulin molecules, which include antibodies having a humanidiotype. In heavy chain loci human or non-human-derived D-gene segmentsmay be included in the artificial Ig loci. The gene segments in suchloci are juxtaposed with respect to each other in an unrearrangedconfiguration (or “the germline configuration”), or in a partially orfully rearranged configuration. The artificial Ig loci have the capacityto undergo gene rearrangement (if the gene segments are not fullyrearranged) in the subject animal thereby producing a diversifiedrepertoire of immunoglobulins having human idiotypes.

Regulatory elements like promoters, enhancers, switch regions,recombination signals, and the like may be of human or non-human origin.What is required is that the elements be operable in the animal speciesconcerned, in order to render the artificial loci functional.

In one aspect, the invention provides transgenic constructs containingan artificial heavy chain locus capable of undergoing gene rearrangementin the host animal thereby producing a diversified repertoire of heavychains having human idiotypes. An artificial heavy chain locus of thetransgene contains a V-region with at least one human V gene segment.Preferably, the V-region includes at least about 5-100 human heavy chainV (or “VH”) gene segments. As described above, a human VH segmentencompasses naturally occurring sequences of a human VH gene segment,degenerate forms of naturally occurring sequences of a human VH genesegment, as well as synthetic sequences that encode a polypeptidesequence substantially (i.e., at least about 85%-95%) identical to ahuman heavy chain V domain polypeptide.

In a preferred embodiment, the artificial heavy chain locus contains atleast one or several rat constant region genes, e.g., Cδ, Cμ and Cγ(including any of the Cγ subclasses).

In another preferred embodiment, the artificial heavy chain locuscontains artificial constant region genes. In a preferred embodiment,such artificial constant region genes encode a human CH1 domain and ratCH2 CH3 domains, or a human CH1 and rat CH2, CH3 and CH4 domains. Ahybrid heavy chain with a human CH1 domain pairs effectively with afully human light chain.

In another preferred embodiment, the artificial heavy chain locuscontains artificial constant region genes lacking CH1 domains In apreferred embodiment, such artificial constant region genes encodetruncated IgM and/or IgG lacking the CH1 domain but comprising CH2, andCH3, or CH1, CH2, CH3 and CH4 domains. Heavy chains lacking CH1 domainscannot pair effectively with Ig light chains and form heavy chain onlyantibodies.

In another aspect, the invention provides transgenic constructscontaining an artificial light chain locus capable of undergoing generearrangement in the host animal thereby producing a diversifiedrepertoire of light chains having human idiotypes. An artificial lightchain locus of the transgene contains a V-region with at least one humanV gene segment, e.g., a V-region having at least one human VL geneand/or at least one rearranged human VJ segment. Preferably, theV-region includes at least about 5-100 human light chain V (or “VL”)gene segments. Consistently, a human VL segment encompasses naturallyoccurring sequences of a human VL gene segment, degenerate forms ofnaturally occurring sequences of a human VL gene segment, as well assynthetic sequences that encode a polypeptide sequence substantially(i.e., at least about 85%-95%) identical to a human light chain V domainpolypeptide. In one embodiment, the artificial light chain Ig locus hasa C-region having at least one rat C gene (e.g., rat Cλ or Cκ).

Another aspect of the present invention is directed to methods of makinga transgenic vector containing an artificial Ig locus. Such methodsinvolve isolating Ig loci or fragments thereof, and combining the same,with one or several DNA fragments comprising sequences encoding human Vregion elements. The Ig gene segment(s) are inserted into the artificialIg locus or a portion thereof by ligation or homologous recombination insuch a way as to retain the capacity of the locus to undergo effectivegene rearrangement in the subject animal.

Preferably, a non-human Ig locus is isolated by screening a library ofplasmids, cosmids, YACs or BACs, and the like, prepared from the genomicDNA of the same. YAC clones can carry DNA fragments of up to 2megabases, thus an entire animal heavy chain locus or a large portionthereof can be isolated in one YAC clone, or reconstructed to becontained in one YAC clone. BAC clones are capable of carrying DNAfragments of smaller sizes (about 50-500 kb). However, multiple BACclones containing overlapping fragments of an Ig locus can be separatelyaltered and subsequently injected together into an animal recipientcell, wherein the overlapping fragments recombine in the recipientanimal cell to generate a continuous Ig locus.

Human Ig gene segments can be integrated into the Ig locus on a vector(e.g., a BAC clone) by a variety of methods, including ligation of DNAfragments, or insertion of DNA fragments by homologous recombination.Integration of the human Ig gene segments is done in such a way that thehuman Ig gene segment is operably linked to the host animal sequence inthe transgene to produce a functional humanized Ig locus, i.e., an Iglocus capable of gene rearrangement which lead to the production of adiversified repertoire of antibodies with human idiotypes. Homologousrecombination can be performed in bacteria, yeast and other cells with ahigh frequency of homologous recombination events. Engineered YACs andBACs can be readily isolated from the cells and used in makingtransgenic animals.

Immunoglobulins Having a Human Idiotype

Once a transgenic animal capable of producing immunoglobulins having ahuman idiotype is made, immunoglobulins and antibody preparationsagainst an antigen can be readily obtained by immunizing the animal withthe antigen. “Polyclonal antisera composition” as used herein includesaffinity purified polyclonal antibody preparations.

A variety of antigens can be used to immunize a transgenic animal. Suchantigens include but are not limited to, microorganisms, e.g. virusesand unicellular organisms (such as bacteria and fungi), alive,attenuated or dead, fragments of the microorganisms, or antigenicmolecules isolated from the microorganisms.

Preferred bacterial antigens for use in immunizing an animal includepurified antigens from Staphylococcus aureus such as capsularpolysaccharides type 5 and 8, recombinant versions of virulence factorssuch as alpha-toxin, adhesin binding proteins, collagen bindingproteins, and fibronectin binding proteins. Preferred bacterial antigensalso include an attenuated version of S. aureus, Pseudomonas aeruginosa,enterococcus, enterobacter, and Klebsiella pneumoniae, or culturesupernatant from these bacteria cells. Other bacterial antigens whichcan be used in immunization include purified lipopolysaccharide (LPS),capsular antigens, capsular polysaccharides and/or recombinant versionsof the outer membrane proteins, fibronectin binding proteins, endotoxin,and exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,and Klebsiella pneumoniae.

Preferred antigens for the generation of antibodies against fungiinclude attenuated version of fungi or outer membrane proteins thereof,which fungi include, but are not limited to, Candida albicans, Candidaparapsilosis, Candida tropicalis, and Cryptococcus neoformans.

Preferred antigens for use in immunization in order to generateantibodies against viruses include the envelop proteins and attenuatedversions of viruses which include, but are not limited to respiratorysynctial virus (RSV) (particularly the F-Protein), Hepatitis C virus(HCV), Hepatits B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

Antibodies specific for cancer can be generated by immunizing transgenicanimals with isolated tumor cells or tumor cell lines as well astumor-associated antigens which include, but are not limited to,Her-2-neu antigen (antibodies against which are useful for the treatmentof breast cancer); CD20, CD22 and CD53 antigens (antibodies againstwhich are useful for the treatment of B cell lymphomas), prostatespecific membrane antigen (PMSA) (antibodies against which are usefulfor the treatment of prostate cancer), and 17-1A molecule (antibodiesagainst which are useful for the treatment of colon cancer).

The antigens can be administered to a transgenic animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

For making a monoclonal antibody, spleen cells are isolated from theimmunized transgenic animal and used either in cell fusion withtransformed cell lines for the production of hybridomas, or cDNAsencoding antibodies are cloned by standard molecular biology techniquesand expressed in transfected cells. The procedures for making monoclonalantibodies are well established in the art. See, e.g., European PatentApplication 0 583 980 A1 (“Method For Generating Monoclonal AntibodiesFrom Rabbits”), U.S. Pat. No. 4,977,081 (“Stable Rabbit-Mouse HybridomasAnd Secretion Products Thereof”), WO 97/16537 (“Stable Chicken B-cellLine And Method of Use Thereof”), and EP 0 491 057 B1 (“Hybridoma WhichProduces Avian Specific Immunoglobulin G”), the disclosures of which areincorporated herein by reference. In vitro production of monoclonalantibodies from cloned cDNA molecules has been described byAndris-Widhopf et al., “Methods for the generation of chicken monoclonalantibody fragments by phage display”, J Immunol Methods 242:159 (2000),and by Burton, D. R., “Phage display”, Immunotechnology 1:87 (1995).

Once chimeric monoclonal antibodies with human idiotypes have beengenerated, such chimeric antibodies can be easily converted into fullyhuman antibodies using standard molecular biology techniques. Fullyhuman monoclonal antibodies are not immunogenic in humans and areappropriate for use in the therapeutic treatment of human subjects.

Antibodies of the Invention Include Heavy Chain-Only Antibodies

In one embodiment, transgenic animals which lack a functional Ig lightchain locus, and comprising an artificial heavy chain locus, areimmunized with antigen to produce heavy chain-only antibodies thatspecifically bind to antigen.

In one embodiment, the invention provides monoclonal antibody producingcells derived from such animals, as well as nucleic acids derivedtherefrom. Also provided are hybridomas derived therefrom. Also providedare fully human heavy chain-only antibodies, as well as encoding nucleicacids, derived therefrom.

Teachings on heavy chain-only antibodies are found in the art. Forexample, see PCT publications WO02085944, WO02085945, WO2006008548, andWO2007096779. See also U.S. Pat. Nos. 5,840,526; 5,874,541; 6,005,079;6,765,087; 5,800,988; EP 1589107; WO 9734103; and U.S. Pat. No.6,015,695.

Pharmaceutical Compositions

In a further embodiment of the present invention, purified monoclonal orpolyclonal antibodies are admixed with an appropriate pharmaceuticalcarrier suitable for administration to patients, to providepharmaceutical compositions.

Patients treated with the pharmaceutical compositions of the inventionare preferably mammals, more preferably humans, though veterinary usesare also contemplated.

Pharmaceutically acceptable carriers which can be employed in thepresent pharmaceutical compositions can be any and all solvents,dispersion media, isotonic agents and the like. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of the antibodiescontained therein, its use in the pharmaceutical compositions of thepresent invention is appropriate.

The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers.Examples of carriers include oils, water, saline solutions, alcohol,sugar, gel, lipids, liposomes, resins, porous matrices, binders,fillers, coatings, preservatives and the like, or combinations thereof.

Methods of Treatment

In a further aspect of the present invention, methods are provided fortreating a disease in a vertebrate, preferably a mammal, preferably aprimate, with human subjects being an especially preferred embodiment,by administering a purified antibody composition of the inventiondesirable for treating such disease.

The antibody compositions can be used to bind and neutralize or modulatean antigenic entity in human body tissues that causes or contributes todisease or that elicits undesired or abnormal immune responses. An“antigenic entity” is herein defined to encompass any soluble or cellsurface bound molecules including proteins, as well as cells orinfectious disease-causing organisms or agents that are at least capableof binding to an antibody and preferably are also capable of stimulatingan immune response.

Administration of an antibody composition against an infectious agent asa monotherapy or in combination with chemotherapy results in eliminationof infectious particles. A single administration of antibodies decreasesthe number of infectious particles generally 10 to 100 fold, morecommonly more than 1000-fold. Similarly, antibody therapy in patientswith a malignant disease employed as a monotherapy or in combinationwith chemotherapy reduces the number of malignant cells generally 10 to100 fold, or more than 1000-fold. Therapy may be repeated over anextended amount of time to assure the complete elimination of infectiousparticles, malignant cells, etc. In some instances, therapy withantibody preparations will be continued for extended periods of time inthe absence of detectable amounts of infectious particles or undesirablecells.

Similarly, the use of antibody therapy for the modulation of immuneresponses may consist of single or multiple administrations oftherapeutic antibodies. Therapy may be continued for extended periods oftime in the absence of any disease symptoms.

The subject treatment may be employed in conjunction with chemotherapyat dosages sufficient to inhibit infectious disease or malignancies. Inautoimmune disease patients or transplant recipients, antibody therapymay be employed in conjunction with immunosuppressive therapy at dosagessufficient to inhibit immune reactions.

All citations are expressly incorporated herein in their entirety byreference.

Experimental

Directed Evolution of homing endonucleases specific for ratimmunoglobulin sequences.

An analysis of rat IgM exon sequences resulted in the identification ofseveral target cleavage sequences for engineered homing endonucleases.Using homing endonuclease I-SceI, two target sequences were identified,one within rat IgM exon II (CGTGGATCACAGGGGTCT) and the other within ratIgM exon III (CTGGGATAACAGGAAGGA). These sites share 61% (11 out of 18bases) sequence identity with the natural recognition sequence of I-SceI(TAGGGATAACAGGGTAAT).

TABLE 1 Target sequences in rat IgM exons(the different nucleotides are underlined) Target Sequence Similarityposition T3 CGTGGATCACAGGGGTCT 61% Exon II T4 CTGGGATAACAGGAAGGA 61%Exon III Wild TAGGGATAACAGGGTAAT type

For the engineering of homing endonucleases specific for these targetsequences we used a highly sensitive selection for the directedevolution of homing endonucleases that couples enzymatic DNA cleavagewith the survival of host cells (described in detail by Chen and Zhao,Nucleic Acid Research 33(18):e154, 2005). An in vitro coevolutionstrategy was used to engineer I-SceI variants with target sequencespecificity. As shown in Table 2, for target sequence T3, two newsequences, T3i1 and T3i2, were selected as intermediate sequences, whilefor target sequence T4, two new sequences, T4i1 and T4i2, were selectedas intermediate sequences. The T3i1 and T4i1 sequences were cloned intothe report plasmid to yield p11-LacY-T3i1 and p11-LacY-T4i1,respectively.

TABLE 2 Sequences in three steps(the different nucleotides are underlined) Step1 T3i1 TAGGGATAA T4i1TAGGGATAA CAGGGGTCT CAGGGAGGA Step2 T3i2 CGTGGATAA T4i2 CTGGGATAACAGGGGTCT CAGGAAGGA Step3 T3 CGTGGATCA T4 CTGGGATAA CAGGGGTCT CAGGAAGGA

To obtain I-SceI mutants with T3i1 or T4i1 sequence specificity,molecular modeling was first carried out to identify the residues to beused to create a focused library via saturation mutagenesis. As shown inFIG. 2, I-SceI binds to the 3′ end of T3i1 or T4i1 through a relaxedloop that lies in the minor groove of DNA. Residues Gly13, Pro14, Asn15and Lys20 are close to this 3′ end and Asn15 binds directly to the lastthymine at the 3′ end of the wild type recognition sequence throughhydrogen bonds. A library of mutants containing all the possiblecombinations of amino acid substitutions at these four select residueswere constructed by saturation mutagenesis. To generate a large enoughlibrary, the ligation reaction and DNA transformation procedures wereoptimized through several trials. A library consisting of 2.9×10⁶mutants was created.

The library was screened for I-SceI mutants with increased activitytowards the T3i1 sequence. Compared to round 0 (wild type I-SceI), thefirst round of screening yielded mutants with increased activity towardthe T3i1 sequence since the cell survival rate was increased by 10-fold.Enrichment of the potentially positive mutants in round 2 and 3 showedfurther improvement in cell survival rate. Similarly, the library wasscreened for I-SceI mutants with increased activity towards the T4i1sequence. Screening of mutants yielded mutants with increased activitytoward the T4i1 sequence.

In parallel, a second library of I-SceI mutants targeting the 5′ end ofthe recognition sequence was designed. The first library created usingsaturation mutagenesis was focused on those residues interacting withthe 3′ end of the four nucleotides of the I-SceI recognition sequence.Based on molecular modeling, Trp149, Asp150, Tyr151 and Asn152 lie inthe major groove formed by the 5′ end nucleotides. Asn152 interactsdirectly with T(−7) though hydrogen bonding. Asp150 and Tyr152 interactT opposite to A(−6) indirectly though a water molecule. Trp149 andTyr151 interact with the phosphate backbone. Thus these four residuesare important to the sequence specificity of I-SceI and simultaneoussaturation mutagenesis on these four residues was done to create asecond I-SceI mutant library.

Further coevolution of these enzymes results in the generation of novelmeganucleases specific for target sequences in rat IgM exons II and III(CGTGGATCACAGGGGTCT and CTGGGATAACAGGAAGGA)

Engineering of I-Cre with Defined Sequence Specificity

For the engineering of homing endonucleases specific for novel targetsequences we used a highly sensitive selection for the directedevolution of homing endonucleases that couples enzymatic DNA cleavagewith the survival of host cells (described in detail by Chen and Zhao,Nucleic Acid Research 33(18):e154, 2005). In addition, a generalstrategy for engineering I-CreI mutant with defined sequence specificitywas designed. I-CreI recognizes a target sequence in a pseudopalindromic manner. Palindromic bases are directly recognized by I-CreIand may be difficult to be altered (J. Mol. Biol., 280, 345-353) (FIG.4).

This property hinders the direct engineering of I-CreI derivatives thatrecognize a non-palindromic sequence. To overcome this problem, thetarget sequence was divided into left-half (upstream-half) andright-half (downstream-half). I-CreI is optimized for the intermediatesequences of the left-half palindrome and the right-half palindrome,respectively (FIG. 4). Then, the I-CreI mutants, optimized forintermediate sequences, are engineered to recognize the target sequencepalindrome. Finally, I-CreI mutant respectively optimized for left-halfand that for right-half will be co-expressed to cleave the targetsequence. In addition, fusion of the left-half optimized mutant with theright-half optimized mutant by a polypeptide linker is examined.

A target sequence within exon IV (CAACTGATCCTGAGGGAGTCGG) that shares59% sequence identity with the natural recognition sequence of homingendonuclease I-CreI was identified. Subsequently, based on the identityof palindromic bases within the original ICreI target sequence, twosequences, T5 and T6, were selected as target sequences for I-CreIengineering.

I-CreI recognition sequence and 2 target sequences:

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 First halfSecond half Homology Orig- A B C D palin- inal C A A A A C G T C G T G AG A C A G T T T G total dromic T5 A A A A A T G T C G T G A G A C A G TT T G 50.0% 64.3% T6 C A A C T G A T C C T G A G G G A G T C G G 59.1%57.1% Palindromic bases are shown in columns A, B, C, and D. Conservedbases are written in bold face.

The two target sequences, T5 and T6, were cloned into reporter plasmids.The I-CreI gene was cloned into the pTrc plasmid and sequenced toconfirm that no mutations were introduced during PCR amplification. TheI-CreI selection system is evaluated for cell survival rates.

In addition, molecular modeling was performed and protein residues thatcontact directly the DNA substrate were identified. In addition, wedesigned the intermediate sequences for in vitro co-evolutionexperiments.

Target residues for saturation mutagenesis Target residue YN-TS5-L Q26and S32 YN-TS5-Ri1 R68, R70 and D75 YN-TS5-Ri2 Q26 and K28 YN-TS5-Ri3N30, Y33 and Q38 YN-TS6-L Q26, K28 and R68 YN-TS6-Ri1 Q44 and R68YN-TS6-Ri2 N30, Y33 and Q38

Subsequently, libraries of ICreI mutants are generated and screened forICreI derivatives with novel target sequences. Further coevolution ofthese enzymes results in the generation of novel meganucleases specificfor a target sequence within exon IV of rat IgM(CAACTGATCCTGAGGGAGTCGG).

Engineering of Zinc-Finger Nucleases

Zinc-finger proteins (ZFP) were designed against sequences encoding ratIgM (exons 1-4) and assembled as described (Zhang, L. et al. Syntheticzing finger transcription factor action at an endogenous chromosomalsite. Activation of the human erythropoietin gene. J. Biol. Chem275:33850-33860, 2000, and Liu, P. Q. et al. Regulation of an endogenouslocus against a panel of designed zinc finger proteins targeted toaccessible chromatin regions. Activation of vascular endothelial growthfactor. J Biol. Chem. 2765:11323-11334, 2001), to yield the followingZFP moieties

Recognition Finger Finger Finger Finger Finger Finger Linker Linker SBSsequence 1 2 3 4 5 6 2-3 4-5 17063 AGACAGGGGGCTCTC NKVGLIE TSSDLSRRSDHLSR RSDNLSE QNAHRKT TGGERP TGEKP 17065 AATTTGGTGGCCATG RSDALSTDRSTRTK RSDALAR RSDSLSA TSSNRKT TGGQRP TGEKP 17067 GTTCTGGTAGTT RSANLARRSDNLRE TSGSLSR QSGSLTR RSDVLSE TGGGGS TGSQKP QRP 17068 GAAGTCATGCAGGGTGDRSALSR TSGHLSR RSDNLST HNATRIN DRSALSR TSGSLTR TGGQRP TGSQKP TC 17089GGTGCCATTGGGGTG RSDALAR RSDHLST HSNARKN ERGTLAR TSGHLSR QSGNLAR TGEKPTGSQKP 17090 GCTGTGGGTGTGGCT QSSDLSR RSDALTQ TSGHLSR RSDALSR DRSDLSRTGGQRP TGEKP 17119 ACCATGTGTGGCAGGG RSAHLSR QSGDLTR RSDALAR RSDTLSVDNSTRIK TGEKP TGEKP 17120 GAGGACCGTGGACAAG RSANLSV DRANLSR RSDALARDRSDLSR RSDDLTR TGEKP TGEKP

DNA encoding ZFPs were cloned into an expression vector. Rat C6 cellswere obtained from the American Type Culture Collection and grown asrecommended in F-12 medium (Invitrogen) supplemented with 5% qualifiedfetal calf serum (FCS, Hyclone), 15% horse serum (Invitrogen) and 5 mMglutamine. Cells were disassociated from plasticware using TrypLE Selectprotease (Invitrogen). For transfection, 200,000 C6 cells were mixedwith 400 ng plamid DNA and 204 Amaxa Solution SF. Cells were transfectedin an Amaxa Nucleofector II Shuttle using program 96 FF-137 andrecovered into 0.1 L warm, supplemented, F-12 medium. Three and ninedays post transfection cells were harvested and chromosomal DNA wasprepared using a Quick Extract Solution 1.0 (Epicentre). The appropriateregion of the IgM locus was PCR amplified using Accuprime High-fidelityDNA polymerase (Invitrogen). PCR reactions were heated to 94°, thengradually cooled to room temperature. Approximately 200 ng of theannealed DNA was mixed with 0.334 CEL-I enzyme (Transgenomic) andincubated for 20 minutes at 42°. Reaction products were analyzed bypolyacrylamide gel electrophoresis in 1×Tris-borate-EDTA buffer. Atypical example demonstrating cleavage activity is shown in FIG. 6.

Generation of Rats with Inactivated Endogenous Heavy Chain Locus UsingExpression Plasmids Encoding a Meganuclease

A cDNA sequence encoding a meganuclease specific for a rat Cμ exon iscloned into an expression vector where expression is controlled by thetetracycline operator sequence. Plasmid DNA is linearized by restrictionenzyme digestion and purified. Rat oocytes are fertilized with spermform rats with a transgene encoding a tetracycline-responsive reversetransactivator. Purified plasmid DNA is injected into pronuclei of suchfertilized rat oocytes. Subsequently, rat embryos are transferred intofoster mothers and brought to term. Newborns are analyzed for thepresence of meganuclease-encoding transgene by PCR using DNA isolatedfrom tissue samples. Male transgenic founder animals are housed for fourmonths when they reach sexual maturity. Expression of meganuclease intransgenic animals is induced by daily administration of doxycycline forone to seven days. Subsequently, sperm is collected twice per week andanalyzed by PCR. Male animals producing mutated sperm are used forbreeding. Offspring with mutated rat Cμ are identified by PCR analysisof tissue samples.

Generation of Rats with Inactivated Endogenous Heavy Chain Locus byMicroinjection of Fertilized Oocytes with Plasmid DNA Encoding aSpecific Meganuclease

A cDNA sequence encoding a meganuclease specific for a rat Cμ exon iscloned into an expression vector where expression is controlled by theCAG-promoter. Purified plasmid DNA is is injected into pronuclei offertilized rat oocytes. Subsequently, rat embryos are transferred intofoster mothers and brought to term. Newborns are analyzed for thepresence mutated IgM exons by PCR and direct sequencing. Alternatively,animals containing cells with mutated IgM exons are identified byincubation of heated and cooled PCR products with CEL-I enzyme andsubsequent gel electrophoresis.

1-34. (canceled)
 35. A chimeric immunoglobulin (Ig) that specificallybinds an antigen, wherein the chimeric Ig is produced by a ratnullizygous for endogenous Ig heavy chain and light chain loci andcomprises: a chimeric heavy chain encoded by an artificial heavy chainlocus that comprises rat and human sequences, including a human variable(V) gene segment; and a light chain encoded by a light chain locuscomprising a human V gene segment and at least one human constant (C)region gene segment; wherein the chimeric Ig has a human idiotype. 36.The chimeric immunoglobulin of claim 35, wherein the chimeric heavychain further comprises a human Ig diversity (D) region gene.
 37. Thechimeric immunoglobulin of claim 36 wherein the chimeric heavy chainfurther comprises a human Ig joining (J) region gene.
 38. The chimericimmunoglobulin of claim 35 wherein the chimeric heavy chain furthercomprises a human C region gene segment.
 39. The chimeric immunoglobulinof claim 35 wherein the light chain is a chimeric light chain comprisingboth rat and human sequences.
 40. A chimeric immunoglobulin (Ig) thatspecifically binds an antigen, wherein the chimeric Ig is produced by arat nullizygous for the endogenous Ig heavy chain locus and comprises achimeric heavy chain encoded by an artificial heavy chain locus thatcomprises rat and human sequences, including a human variable (V) genesegment, wherein the chimeric Ig has a human idiotype.
 41. The chimericimmunoglobulin of claim 40 wherein the chimeric heavy chain furthercomprises a human Ig diversity (D) region gene.
 42. The chimericimmunoglobulin of claim 41 wherein the chimeric heavy chain furthercomprises a human Ig joining (J) region gene.
 43. The chimericimmunoglobulin of claim 40 wherein the chimeric heavy chain furthercomprises a human constant (C) region gene segment.